A variety of sequence-specific processes have been developed that make a specific, directed genetic alteration in a cultured cell. The desired alteration most often is a nucleotide mutation, for example to correct a genetic defect or to introduce an in-frame stop codon and thereby “knock-out” the target gene. The methods have in common the step of introducing into the cells of the culture an exogenous nucleic acid having the desired sequence, i.e., the exogenous nucleic acid “encodes” the desired mutation. The exogenous nucleic acid can be a duplex “hairpin” “chimeric” oligonucleotide of between about 40 and 100 nucleotides including 2′ alkoxy substituted ribonucleotides (Cole-Strauss, et al., 1996, Science 273, 1386-89), an end-protected olignucleotide (WO 01/15740; Gamper et al., 2000, NAR 28, 4332-39) or unprotected DNA fragments of between about 100 and 2000 nucleotides, which can be optionally separated so that the introduced nucleic acid is substantially free of either the sense or antisense strand. Goncz et al., 1998, Hum. Mol. Genetics 7, 1913; Kunzelmann et al., 1996, Gene Ther. 3, 859; U.S. Pat. No. 6,010,908. The exogenous nucleic forms a duplex with the homologous region of the genomic DNA (the “target genomic fragment”) and the cell's enzymatic machinery causes the desired mutation in the target genomic fragment.
Chimeric hairpin oligonucleotides can be used to mutate plant cells. Beetham et al., 1999, Proc. Natl. Acad. Sci. 96, 8774; Zhu et al., 1999, Proc. Natl. Acad. Sci. 96, 8768; WO 98/54330; WO 99/07865; WO 99/07865.
A sequence-specific process to induce mutations in yeast using phosphorothioate end-protected single stranded oligonucleotides has been developed and 2′O-4′ methylene bl;ocked oligonucleotides. Parekh-Olmedo, H., et al., 2002, Chem. Biol. 9, 1073-84; Liu, L., et al., 2002, NAR 30, 2742-50; Liu, L., et al., 2002, Mol. Cell. Biol. 22, 3852-63.
A problem that has limited the use of sequence-specific processes is that the fraction of the cultured cells that contain the desired mutation can be very small. Under these circumstances there is no practical way to identify and clone the altered cells unless the desired alteration confers some selectable phenotype, such as drug resistance, or a grossly visible phenotype that permits cloning by inspection.
Techniques have been developed to permit the detection of single nucleotide mutation in cultured cells. One common technique is allele specific polymerase chain reaction (AS-PCR). PCR is the technique whereby two primers are used to amplify a template sequence using bacterial enzymes in a cell free system. The DNA polymerase employed in PCR requires that the primer be hybridized (Watson-Crick paired) to the template DNA for synthesis to occur. Therefore, if the hybridization conditions are made sufficiently stringent, a single nucleotide mismatch between template and one of the two primers can cause a readily detectable difference in the amount of DNA that is synthesized in the PCR process. This technique permits the use of allele specific primers to distinguish the genotype of a homogeneous DNA sample from an allelic genotype that differs by a single nucleotide mutation. Particular attention has been drawn to the effects of mismatches at the 3′ end of the primer. Reviewed, Bottema, C. D., & Sommer, S. S., 1993, Mutation Research 288, 93-102.
However, AS-PCR is a reportedly satisfactory method to detect a rare cell of one genotype in the presence of the allelic genotype only up to a sensitivity of 1 in 100. Kirby, G. M., et al., 1996, Int. J. Cancer 68, 21-25. Experience has shown that when the hybridization stringency is high enough to suppress amplification of the unwanted allele, i.e., to prevent false positives, AS-PCR becomes insensitive to the presence of the rare cell having the correct allele.
In addition to AS-PCR, other techniques have been developed to readily detect single nucleotide differences in small samples of DNA. The oldest is the restriction enzyme technology that is used to detect restriction fragment length polymorphism (RFLP). Restriction enzymes are DNA endonucleases that cut the DNA polymer whenever they encounter a specific nucleotide sequence that is typically a palindrome between 4 and 8 nucleotides in length. Other techniques include direct sequencing, in particular, the technique called “pyrosequencing” uses a luciferase-based detection of the production of pyrophosphate (a phosphoric acid anhydride), which occurs during DNA polymerization. Ronaghi et al., 1998, Science 281, 363; Ronaghi et al. 2000, Anal. Biochem. 286, 282-8; U.S. Pat. No. 6,210,891. Pyrosequencing has been shown to be effective in detecting a single nucleotide difference in as few as one in 20 cells, but not fewer. Hochberg, E. P., et al., 2002, entitled: “A novel rapid single nucleotide polymorphism based method for assessment of hematopoietic chimerism” Blood [e-published at www.bloodjouurnal.org].
There remains a need for a method of detecting mutant cells at frequencies less than one in 20.